Aspirin Effects and Aspirin Resistance
Aspirin is known to reduce post-acute myocardial infarction (AMI) cardiovascular events by 25-30%. Recent findings indicate that the incidence of aspirin non-responders is between 5-40% of the treated population. It has been proposed that this sub-population of patients does not benefit from aspirin treatment and has an increased risk for future cardiovascular events. Although bedside devices exist for monitoring platelet function, these devices were not designed to specifically examine aspirin non-responsiveness. Therefore, the real prevalence of aspirin non-responders remains unknown. Moreover, existing bedside platelet monitoring devices are affected by platelet P2 receptor P2Y12 antagonism. P2Y12 antagonism induced by PLAVIX™ which is commonly prescribed for patients with acute coronary syndromes (ACS). Since this patient population is at high risk for thrombotic events, and has been found to have a high incidence of aspirin non-responsiveness, and since PLAVIX™ requires aspirin co-therapy to be maximally effective, effective management of this patient population requires that new platelet monitoring devices and methods be employed.
One method for detecting aspirin resistance in the background of PLAVIX™ use comprises the step of evaluating simultaneously in real time the effect of aspirin on thrombus formation triggered by a collagen-coated capillary and by arachidonic acid-induced whole blood thrombus formation under arterial shear rates.
The antithrombotic properties of aspirin were reported more than 50 years ago and are mostly attributed to the inhibition of prostaglandin synthesis. Aspirin treatment is known to exhibit both anti-thrombotic and anti-inflammatory activities, thought to be mediated by irreversible acetylation of Ser530 of cycloxygenase type 1 (Cox-1) and Ser516 of cycloxygenase type 2 (Cox-2), respectively. Cox-1 is constitutively expressed whereas Cox-2 is inducible. This is of particular importance when evaluating the anti- and potential pro-thrombotic activities of aspirin. As platelets are anucleate cells, aspirin inhibition of the synthesis of the pro-aggregatory and vasoconstrictor metabolite thromboxane A2 (TxA2) lasts for the life of the cell even though aspirin's half life is only 20 minutes. By comparison, aspirin inhibition of Cox-2, and the subsequent prevention of prostacyclin (PGI2) by endothelial cells is rapidly overcome by newly synthesized Cox-2. Resynthesis of Cox-2 is judged to be beneficial as PGI2 is antiaggregatory and vasodilatory. Benefits of aspirin therapy in cardio/cerebrovascular diseases are well documented. For example, aspirin is known to reduce the incidence of AMI in the acute phase of unstable angina by up to 25% (Lancet, 2(8607):349-60 (1988); Bmj, 308(6921):81-106 (1994)), and to reduce mortality and recurrent stroke in patients with acute ischemic stroke (Lancet, 349(9065):1569-81 (1997)). A meta-analysis recently suggested that the use of aspirin in patients population with diabetes and peripheral arterial disease be expanded (Bmj 324(7329):71-86 (2002)).
However, in the past 15 years there have been several studies reporting the existence of a sub-population of patients resistant to the antithrombotic activity of aspirin. The prevalence of aspirin resistance is reported to range between 5 and 40%. The broad range is attributed to the absence of a reliable assay that will specifically assess the antithrombotic properties of aspirin.
Several hypotheses have been proposed for aspirin resistance, some of which concern aspirin, others not: Cox-2 expression in platelets, Cox-2 activation in inflammatory and vascular cells, and/or production of eicosanoids, increased reaction to collagen or adenosine diphosphate (ADP), presence of erythrocytes, interaction between aspirin and non-steroidal anti-inflammatory drugs (NSAIDs), variant isoforms of Cox-1, increased platelet turnover, poor compliance, increased amount of plasma von Willebrand factor (vWF), or genetic polymorphisms (of glycoprotein IIb-IIIa (GP IIb-IIIa), GP Ia-IIa, and eventually GP Ibα). Several studies have shown that aspirin resistance may be inducible. For example, one study showed that about 30% of people become resistant while under chronic aspirin therapy (Helgason, C. M. et al., Stroke, 25(12):2331-6 (1994)) and another showed that an increase in percentage of aspirin-resistant patients occurs following surgical procedures (i.e. post coronary artery bypass grafting (CABG), Zimmermann, N. et al., J Thorac Cardiovasc Surg, 121(5):982-4 (2001)). Others have reported a progressive reduction in platelet sensitivity to aspirin as measured by platelet aggregation in long-term treated patients (Pulcinelli, F. M. et al., J Am Coll Cardiol, 43(6):979-84 (2004)). Moreover, the lack of reliable tools that specifically measure aspirin effects may directly contribute to the extent of the aspirin resistance phenomenon. Indeed, aspirin resistance may be also dose-dependent, and it is plausible that part of the aspirin resistant population could benefit from a personalized therapy. The development of an assay that will allow quantitative assessment of these issues is therefore necessary.
The clinical consequences of aspirin resistance are of major importance as it is now commonly accepted that it correlates with future cardiovascular events (Gum, P. A. et al., J Am Coll Cardiol, 41(6):961-5 (2003); Eikelboom, J. W. et al., Circulation, 105(14):1650-5 (2002)). An example of aspirin resistance that may correlate with thrombotic events is the interaction of aspirin with NSAIDs. It has been reported that the aspirin-dependent inhibition of platelet aggregation and serum TxB2 formation (stable metabolite of TxA2) was compromised when ibuprofen was administered prior to aspirin (Catella-Lawson, F. et al., N Engl J Med, 345(25):1809-17 (2001)). The binding of ibuprofen is proposed to block the S530 site of Cox-1before its irreversible acetylation by aspirin. This important finding appears to correlate with clinical events as a recent study has highlighted an increased cardiovascular mortality in patients combining ibuprofen plus aspirin vs aspirin alone (MacDonald, T. M. et al., Lancet, 361(9357):573-4 (2003)). The importance of aspirin resistance is further emphasized by the state of the art combination aspirin/P2Y12 inhibition (PLAVIX™), demonstrated to confer higher antithrombotic efficacy than single therapies alone.
A clear, unequivocal definition of aspirin resistance becomes crucial as it could ultimately lead to a personalized antithrombotic strategy. The need for individualized screening is reinforced by the fact aspirin causes gastro-intestinal (GI) and bleeding complications. The GI toxicity of aspirin appears to be dose-dependent, starting with doses as low as 10 mg/day. Since other platelet-inhibiting and anticoagulant agents potentiate the gastro-intestinal lesions and bleeding risk associated with low-dose aspirin, one can therefore question the use of aspirin in aspirin-resistant patients. Moreover, aspirin's side effects are not limited to GI and bleeding complications. Aspirin is thought to induce asthma in as much as 20% of the patients (Jenkins, C. J. et al., Bmj, 328(7437):434 (2004)). This situation is rendered even more paradoxical as a high percentage of patients with aspirin-induced asthma also take NSAIDs (notably ibuprofen).
Laboratory Methods to Detect Aspirin Resistance:
Several laboratory tests of platelet function have been designed and are available to “diagnose” aspirin-resistance using whole blood. Most certainly, the 2 main tools utilized are the Ultegra Rapid Platelet Function Assay (RPFA-ASA), and the PFA-100 device.
The RPFA-ASA cartridge has been specifically designed to address the level of inhibition of platelet aggregation achieved by aspirin treatment. As mentioned by the manufacturer, it is a qualitative measure of the effects of aspirin. In that assay, fibrinogen-coated beads agglutinate platelets through binding to GP IIb-IIIa receptors following stimulation by metallic cations and propyl gallate. The change in optical signal triggered by the agglutination (light transmittance increases as activated platelets bind and agglutinate the beads in the whole blood suspension) is measured. A recent study has detected a high incidence (23%) of aspirin non-responsiveness using this device, and determined a history of coronary artery disease to be associated with twice the odds of being an aspirin non-responder (Wang, J. C. et al., Am J Cardiol, 92(12):1492-4(2003)). Aspirin resistance cannot be evaluated by the RPFA assay, however, in patients who were prescribed either GP inhibitors, dipyridamole, PLAVIX™ (or ticlid), or NSAIDS (ibuprofen, naproxen, diclofenac, indomethacin, piroxicam) since those compounds interfere with the assay.
In the PFA-100 device, the platelet hemostatic capacity (PHC) of a citrated blood sample is determined by the time required for a platelet plug to occlude a 150 μM aperture cut into a collagen-epinephrine coated membrane (used for the detection of aspirin). In the PFA-100 system, samples of citrated blood are aspirated through the aperture at shear rates of ˜4,000-5,000/sec. Under these high conditions of shear, vWF interactions with both GP Ibα and GP IIb-IIIa trigger the thrombotic process. In the context of clinical events, plasma levels of vWF are expected to increase following platelet-rich thrombi formation and endothelial cell injury. Interestingly, Chakroun et al. reported that the aspirin-resistant population measured by PFA-100 also demonstrated an increased plasma vWF ristocetin cofactor activity (Chakroun, T. et al., Br J Haematol, 124(1):80-5 (2004)). Furthermore, a poor inhibition of thrombotic events has been reported for shear rates around 10,000/sec with aspirin (Barstad, R. M. et al., Thromb Haemost, 75(5):827-32 (1996)), and PFA-100 detects desmopressin (DDAVP) therapy which increases plasma levels of vWF (Fressinaud, E. et al., Br J Haematol, 106(3):777-83 (1999)). One can therefore argue that the high incidence of aspirin resistance found post-AMI with the PFA-100 device (Gum, P. A. et al., Am J Cardiol, 88(3):230-5 (2001)) reflects an increase in platelet sensitivity towards high shear induced collagen-vWF/GP Ibα-/GP IIb-IIIa-interactions (Chakroun, T. et al., Br J Haematol, 124(1):80-5 (2004)) rather than true aspirin resistance. In addition, several investigators have found that most of the aspirin-resistant population identified by PFA-100 appeared to be aspirin sensitive as shown by inhibition of platelet aggregation induced by arachidonic acid (AA) (Gum, P. A. et al., J Am Coll Cardiol, 41(6):961-5 (2003); Chakroun, T. et al., Br J Haematol, 124(1):80-5 (2004)), as well as extremely low TxB2 levels (Andersen, K. Thromb Res, 108(1):37-42 (2002)). Moreover, investigators have reported a good prognosis for long term clinical events in the aspirin-resistant population revealed by AA-induced platelet aggregation, but this was not the case for the PFA-100 device. The data described herein also show that the PFA-100 device does not specifically reveal aspirin effects.
Other models have been utilized for examining aspirin effects, such as platelet aggregation and bleeding time. For example, arachidonic acid-induced platelet aggregation in platelet rich plasma is classically considered the gold standard for evaluation of aspirin effects on platelets. Nevertheless, determining aspirin resistance should be investigated using whole blood for several reasons. First, erythocytes may contribute to aspirin-resistance. Valles et al. (Valles, J. et al., Blood, 78(1):154-62 (1991)) have demonstrated that the presence of erythrocytes directly affects platelet reactivity by increasing TxB2 synthesis, release of serotonin (5-HT), β-thromboglobulin (β-TG) and ADP. This has a major impact on the evaluation of aspirin resistance, as the same authors found that aspirin treatment (200-300 mg daily) failed to block platelet reactivity in presence of erythrocytes in two thirds of a group of patients with ischemic heart disease and ischemic stroke, despite full inhibition of TxA2 synthesis (Valles, J. et al., Circulation, 97(4):350-5 (1998)).
Second, nucleated cells (leukocytes) may contribute to aspirin-resistance. It is commonly accepted that because platelets are anucleated cells, their potential for producing TxA2 is irreversibly suppressed during their lifetime after aspirin treatment. However, a potential source of aspirin-insensitive TxA2 and 8-epi-prostaglandin-F 2α (8-epi PGF2α) (2 known platelet agonists) exists as the de novo synthesis of Cox-2 and full recovery of Cox activity occurs 2 to 4 hours after aspirin treatment via stimulated nucleated cells (Maclouf, J. G. et al., Thromb Haemost, 79(4):691-705 (1998)). Platelet microparticles can also indirectly participate in generation of Cox-2-dependent TxA2 formation via delivery of AA by sPLA2. Similarly, it has been reported that AA derived from neutrophils can increase TxA2 formation in platelets (Maugeri, N. et al., Blood, 80(2):447-51 (1992)).
Third, plasma proteins and lipids may contribute to aspirin-resistance. The presence of F2-isoprostanes in plasma are increased in patients with unstable angina, diabetes mellitus, hypercholesterolemia and cigarette smokers. These sub-populations present some of the highest percentage of aspirin-resistant patients. One candidate for mediating aspirin resistance is the 8-epi-PGF2, or isoprostane 8-epi prostaglandin F2α (8-iso-PGF2α). It is produced from AA by nonenzymatic lipid peroxidation catalyzed by oxygen free radicals, and is aspirin-insensitive (Wang, Z. et al., J Pharmacol Exp Ther, 275(1):94-100 (1995)). Consequently, 8-epi-PGF2α, generation can occur in all cellular players of atherosclerosis. It is a potent vasoconstrictor and has been reported to activate platelets at high concentrations and cause dose-dependent irreversible platelet aggregation in the presence of subluminal concentrations of collagen, ADP or arachidonic acid. In addition, 8-epi-PGF2α could potentiate the expression of GP IIb-IIIa and enhance platelet adhesion on fibrinogen. Although a direct interaction of 8-epi-PGF2α with the TPα receptor has been shown, the biological significance of 8-epi-PGF2α in thrombosis remains controversial as low concentrations (such as those found in vivo) may inhibit collagen-induced platelet aggregation but amplify low dose ADP-induced platelet aggregation (Yin, K. et al., J Pharmacol Exp Ther, 270(3): 1192-6 (1994)).
Whole blood platelet aggregation and whole blood aggregation utilizing a screen filtration pressure method have been shown to detect the effects of aspirin. However, these are labor intensive techniques and the evaluation of the aggregation process is performed under low shear rate conditions (like those encountered in veins) which do not reflect the thrombotic process occurring in moderately stenosed arteries.
Template bleeding times have been demonstrated to be accurate in determining platelet function prior to and following ASA administration. This technique is sensitive enough to diagnose platelet dysfunction (notably von Willebrand Disease), but bleeding time is highly variable, and sensitive to all anti-platelet agents, anti-coagulants, and other factors such as alcohol and green tea.
Altogether, the different techniques are either time-consuming, or of poor prognostic for true aspirin resistance. This demonstrates the need for a new device and methods that will specifically evaluate aspirin effects.